New Alleles of <i>SIR2</i> Define Cell-Cycle-Specific Silencing Functions

Matecic, Mirela; Martins-Taylor, Kristen; Hickman, Merrit; Tanny, Jason C.; Moazed, Danesh; Holmes, Scott G.

doi:10.1534/genetics.106.055491

Cited by 19 publications

(23 citation statements)

References 66 publications

(94 reference statements)

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“…Their mutants fell into two classes. One group had defects in deacetylase activity and the other had defects in binding to Sir4 (Matecic et al 2006). Similar to the sir2-276 mutant described here, they found that the deacetylase-defective mutants lost silencing rapidly when raised to the nonpermissive temperature.…”

Section: Discussionsupporting

confidence: 77%

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A Yeast Sir2 Mutant Temperature Sensitive for Silencing

Wang

Landry²,

Sternglanz

2008

Genetics

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A screen for Saccharomyces cerevisiae temperature-sensitive silencing mutants identified a strain with a point mutation in the SIR2 gene. The mutation changed Ser276 to Cys. This amino acid is in the highly conserved NAD 1 binding pocket of the Sir2 family of proteins. Haploid strains of either mating type carrying the mutation were severely defective at mating at 37°but normal at 25°. Measurements of RNA from the HMR locus demonstrated that silencing was lost rapidly upon shifting the mutant from the low to the high temperature, but it took .8 hours to reestablish silencing after a shift back to 25°. Silencing at the rDNA locus was also temperature sensitive. On the other hand, telomeric silencing was totally defective at both temperatures. Enzymatic activity of the recombinant wild-type and mutant Sir2 protein was compared by three different assays. The mutant exhibited less deacetylase activity than the wild-type protein at both 37°and 25°. Interestingly, the mutant had much more NAD 1 -nicotinamide exchange activity than wild type, as did a mutation in the same region of the protein in the Sir2 homolog, Hst2. Thus, mutations in this region of the NAD 1 binding pocket of the protein are able to carry out cleavage of NAD 1 to nicotinamide but are defective at the subsequent deacetylation step of the reaction.

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Section: Discussionsupporting

confidence: 77%

“…Apparently, more Sir2 activity is required in vivo at 37°than at 25°. Holmes and colleagues recently described several sir2 ts mutants generated by error-prone mutagenesis (Matecic et al 2006;Hickman et al 2007). All but one of these mutants had multiple amino acid changes.…”

Section: Discussionmentioning

confidence: 99%

A Yeast Sir2 Mutant Temperature Sensitive for Silencing

Wang

Landry²,

Sternglanz

2008

Genetics

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“…Note that when HML␣ is derepressed in a MATa strain (e.g., by disrupting the SIR genes), the a1 and ␣2 proteins form a complex that negatively regulates the expression of ␣1 and ␣2 (16,26,35). However, ␣1 mRNA is detectable in such a strain, especially when methods with high sensitivity like qRT-PCR are used (9,23,40). Matecic et al has demonstrated that the transcriptional state of HML could be examined quantitatively by measuring the abundance of HML␣1 message (23).…”

Section: Resultsmentioning

confidence: 99%

“…However, ␣1 mRNA is detectable in such a strain, especially when methods with high sensitivity like qRT-PCR are used (9,23,40). Matecic et al has demonstrated that the transcriptional state of HML could be examined quantitatively by measuring the abundance of HML␣1 message (23). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Asymmetric Positioning of Nucleosomes and Directional Establishment of Transcriptionally Silent Chromatin by Saccharomyces cerevisiae Silencers

Zou

2006

Molecular and Cellular Biology

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In Saccharomyces cerevisiae, silencers flanking the HML and HMR loci consist of various combinations of binding sites for Abf1p, Rap1p, and the origin recognition complex (ORC) that serve to recruit the Sir silencing complex, thereby initiating the establishment of transcriptionally silent chromatin. There have been seemingly conflicting reports concerning whether silencers function in an orientation-dependent or -independent manner, and what determines the directionality of a silencer has not been explored. We demonstrate that chromatin plays a key role in determining the potency and directionality of silencers. We show that nucleosomes are asymmetrically distributed around the HML-I or HMR-E silencer so that a nucleosome is positioned close to the Abf1p side but not the ORC side of the silencer. This coincides with preferential association of Sir proteins and transcriptional silencing on the Abf1p side of the silencer. Elimination of the asymmetry in nucleosome positioning at a silencer leads to comparable silencing on both sides. Asymmetric nucleosome positioning in the immediate vicinity of a silencer is independent of its orientation and genomic context, indicating that it is the inherent property of the silencer. Moreover, it is also independent of the Sir complex and thus precedes the formation of silent chromatin. Finally, we demonstrate that asymmetric positioning of nucleosomes and directional silencing by a silencer depend on ORC and Abf1p. We conclude that the HML-I and HMR-E silencers promote asymmetric positioning of nucleosomes, leading to unequal potentials of transcriptional silencing on their sides and, hence, directional silencing.The establishment of condensed and transcriptionally silent heterochromatin in the eukaryotic genome is achieved via an initiation process that recruits silencing/repressor complexes to specific nucleation sequences, followed by their propagation along the chromatin. A silencing complex usually contains a histone-modifying enzyme(s) responsible for yielding heterochromatin-specific "histone codes" and structural proteins that recognize these codes and bind the modified histones with high affinity (13). In the yeast Saccharomyces cerevisiae, transcriptional silencing of the HML and HMR loci as well as regions near the telomeres is mediated by a silent chromatin that shares many similarities with metazoan heterochromatin at the molecular level (13). Silent chromatin at the HM loci is established by combined actions of cis-acting DNA elements and trans-acting proteins (32). The cis-acting elements are small specialized sequences, known as the E and I silencers, that flank the HM loci (Fig. 1A). Each silencer is composed of a unique combination of binding sites for proteins Abf1p, Rap1p, and ORC (for "origin recognition complex for DNA replication") (Fig. 1A), whose binding is required for silencing (32). The trans-acting factors include silencer-binding proteins, histones, and Sir1p through Sir4p. Sir2p is an NAD-dependent histone deacetylase that is believed to be resp...

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“…For instance, a trans-activator is better able to activate a gene repressed by telomeric heterochromatin when expressed at the start of M-phase than it is in G 1 (Aparicio and Gottschling 1994). In addition, specific conditional alleles of the SIR2 silencing factor gene cause an M-phase-specific silencing defect (Matecic et al 2006). Most significantly, the establishment of silencing in yeast requires progression through the cell cycle (Miller and Nasmyth 1984;Fox et al 1997;Kirchmaier and Rine 2001;Li et al 2001;Lau et al 2002;Kirchmaier and Rine 2006).…”

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confidence: 99%

H2A.Z (Htz1) Controls the Cell-Cycle-Dependent Establishment of Transcriptional Silencing at Saccharomyces cerevisiae Telomeres

Martins-Taylor

Sharma²,

Rozario³

et al. 2011

Genetics

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The establishment of transcriptional silencing in Saccharomyces cerevisiae requires progression through the cell cycle. We have previously found that transit through M-phase is necessary and sufficient to establish silencing at telomeres following induction of the Sir3 silencing factor. In this study we find that halting cell-cycle progression in either G 1 or at the beginning of M-phase limits the ability of Sir3 to associate with a telomere-linked reporter gene and prevents the changes in histone modifications associated with gene repression. Deletion of genes coding for the histone variant H2A.Z (Htz1 in yeast) and histone acetyltransferase Sas2 abolish the cell-cycle progression requirement for the establishment of silencing. Cells blocked in telophase (but not at metaphase) are also able to establish silencing. We show that H2A.Z binds to the promoter of our telomere-linked reporter gene and that this binding diminishes in silenced cells. Finally, we observe a specific displacement of H2A.Z from chromatin in telophaseblocked cells, regardless of the silencing status of the reporter gene. These results suggest that the requirement for M-phase in the establishment of silencing may reflect a cell-cycle regulated relaxation of heterochromatin barriers.

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New Alleles of SIR2 Define Cell-Cycle-Specific Silencing Functions

Cited by 19 publications

References 66 publications

A Yeast Sir2 Mutant Temperature Sensitive for Silencing

A Yeast Sir2 Mutant Temperature Sensitive for Silencing

Asymmetric Positioning of Nucleosomes and Directional Establishment of Transcriptionally Silent Chromatin by Saccharomyces cerevisiae Silencers

H2A.Z (Htz1) Controls the Cell-Cycle-Dependent Establishment of Transcriptional Silencing at Saccharomyces cerevisiae Telomeres

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